Liposomes are unilamellar or multilamellar lipid vesicles which enclose a three-dimensional space. The membranes of liposomes are formed by a bimolecular layer of one or more lipid components having polar heads and non-polar tails. In an aqueous (or polar) solution, the polar heads of one layer orient outwardly to extend into the aqueous, or polar, solution and to form a continuous, outer surface. Unilamellar liposomes have one such bimolecular layer, whereas multilamellar vesicles generally have a plurality of substantially concentric bimolecular layers arranged rather like an onion.
Liposomes are well recognized as useful for encapsulating therapeutic agents, such as cytotoxic drugs or other macromolecules capable of modifying cell behavior, and carrying these agents to in vivo sites. For example, U.S. Pat. No. 3,993,754, inventors Rahman et al., issued Nov. 23, 1976, discloses an improved method for chemotherapy of malignant tumors in which an antitumor drug is encapsulated within liposomes and the liposomes are injected into an animal or man. U.S. Pat. No. 4,263,428, inventors Apple, et al., issued Apr. 21, 1981, discloses an antitumor drug which may be more effectively delivered to selective cell sites in a mammalian organism by incorporating the drug within uniformly sized liposomes. Drug administration via liposomes can have reduced toxicity, altered tissue distribution, increased drug effectiveness, and an improved therapeutic index.
Liposomes have also been used in vitro as valuable tools to introduce various chemicals, biochemicals, genetic material and the like into viable cells, and as diagnostic agents.
A variety of methods for preparing liposomes are known, many of which have been described by Szoka and Papahadjopoulos, Ann. Rev. Biophysics Bioeng. 9: 467-508 (1980). Also, several liposome encapsulation methods are disclosed in the patent literature.
For example, U.S. Pat. No. 4,235,871, inventors Papahadjopoulos and Szoka, issued Nov. 25, 1980, describes a method whereby large unilamellar vesicles can be formed which encapsulate large macromolecules. A principle disadvantage of this method is the exposure of the material to be encapsulated to organic solvent, such as diethyl ether, which may result in denaturation of sensitive proteins.
U.S. Pat. No. 4,016,100, inventors Suzuki et al., issued Apr. 5, 1977, describes the entrapment of certain pharmaceuticals in lipid vesicles by freezing the aqueous phospholipid dispersion of pharmaceutical and lipid. It is not clear as to the bio-availability of the total material encapsulated, and the technique may not be efficient for pharmaceuticals of a relatively polar nature. Also, the necessity for freezing, thawing and then separating large volumes is expensive for large-scale, commercial preparation.
Although encapsulation of therapeutic agents and biologically active compounds in liposomes has significant potential for delivering such materials to targeted sites in the human body and for diagnostic applications, producing encapsulated materials on a commercially feasible scale has been a problem. The current methods involve organic solvents or detergents which are expensive, difficult to remove, or present health hazards, and which may interact unfavorably with the therapeutic agents or biologically active molecules to be encapsulated.
It is an object of the present invention that a method be provided which is suitable for the encapsulation of a wide variety of materials, including biologically active macromolecules such as nucleic acids, polypeptides, and enzymes, and which has trapping efficiencies up to about fifty percent of the original material utilized for encapsulation.
It is a further object of the present invention to provide a method which is simple, avoids the use of organic solvents or detergents, and which is feasible and inexpensive for large-scale, commercial production of liposomes having materials encapsulated therein.